WEDNESDAY, OCTOBER 9
ABSOLUTE QUANTIFICATION CONT.
8:25 am Chairperson's Remarks
Donna C. Sullivan, Ph.D., Professor of Medicine, Division of Infectious Diseases, University of Mississippi Medical Center
8:30 Detection of Methicillin-Resistant Staphylococcus Aureus (MRSA) by A Duplex Digital Droplet Polymerase Chain Reaction
We employed a digitaldroplet PCR (ddPCR) end point assay for high-precision MRSA detection. A magnetic extraction and lysis protocol to rapidly process samples from nasal swabs was developed.The ddPCR technology was compared to culture methods and two real-time PCR platforms for detection of MRSA. The ddPCR assay distinguished MRSA from MSSA and other organisms as well as identified MRSA isolates of various genetic backgrounds. The results are the first demonstration of the ddPCR approach for sensitive and specific detection of MRSA.
9:00 dPCR Applications in the Diagnostic Virology Laboratory
Keith R. Jerome, M.D., Ph.D., Professor and Head, Virology Division, Department of Laboratory Medicine, University of Washington; Associate Member, Fred Hutchinson Cancer Research Center, Vaccine and Infectious Disease Division
Digital PCR (dPCR) allows sensitive and accurate absolute quantitation of a nucleic acid sample without the need for a standard curve. While dPCR is still mainly used in research settings, the characteristics of dPCR also make it attractive in the clinical laboratory. In this talk we will discuss applications of dPCR in the diagnostic virology laboratory, including ultraprecise cytomegalovirus monitoring, diagnosis of chromosomally integrated human herpesvirus 6, and quantitation of latent HIV reservoirs.
9:30 Application of Droplet Digital PCR for the Investigation of the Latent HIV Reservoir
Douglas D. Richman, M.D., Director, Center for AIDS Research; Distinguished Professor, Pathology and Medicine; Florence Seeley Riford Chair in AIDS Research, University of California, San Diego
The latent reservoir of HIV represents the obstacle to curing the infection with the currently highly effective antiretroviral therapy. Measuring the reservoir requires assays of rare events in a large number of cells. We have developed and applied droplet digital PCR the assay of various species of HIV DNA and RNA transcripts. The characterization of these assays and their application to clinical studies will be discussed.
10:00 A Novel HIV Therapeutic Approach: Immune Reconstitution in HIV-Infected Subjects by Infusion of Zinc Finger Nuclease Mediated CCR5 Modified CD4+ T-Cells
Gary Lee, Ph.D., Scientist, Team Leader, Sangamo BioSciences
CCR5 is a major co-receptor for HIV entry. The natural delta-32 mutation has demonstrated the benefit of CCR5 deficiency in the context of resistance to HIV infection. SB-728-T is a ZFN mediated CCR5 knockout autologous CD4 T-cell product for treatment of HIV. In a Phase I clinical study, nine chronically HIV infected subjects on long term ART were treated. A single infusion of SB-728-T leads to long term CD4 reconstitution. CCR5 modification is also sustained for the duration of observation. The level of CD4 reconstitution and gene marking is significantly greater than previous T-cell therapies in HIV. More importantly, improvement of CD4 count is associated with significant reduction of HIV reservoir (0.6 log 1 yr post infusion). This result has significant implication in HIV cure research.
10:30 Coffee Break with Exhibit and Poster Viewing
Implementing Digital PCR in the Lab
11:15 VENDOR PANEL: Implementing Digital PCR in the Lab
Moderator: Ross Haynes, Biological Science Technician, Biochemical Science Division, National Institute of Standards and Technology
Panelists: Nicholas Heredia, Ph.D., Staff Scientist, Digital Biology Center, Bio-Rad Laboratories
Andy Watson, Chief Commercial Officer, Raindance Technologies, Inc.
Sabita Sankar, Ph.D., Associate Director, Scientific Affairs, MolecularMD
Stephen Jackson, Ph.D., Associate Director, Product Applications, Life Technologies
12:15 pm Luncheon Presentations (Sponsorship Opportunities Available)or Lunch on Your Own
EMERGING DIGITAL DETECTION TECHNOLOGIES
1:15 SlipChip for Digital Nucleic Acid Amplification
Feng Shen, Director, Research and Development, SlipChip LLC
SlipChip is a microfluidic platform that can generate a large number of reaction compartments by a simple slipping step. The platform has been demonstrated with digital PCR, digital RT-PCR and digital isothermal amplification. Multiplex digital RT-PCR with large dynamic range has also been demonstrated on a multivolume SlipChip for quantitative analysis of nucleic acid, specifically HIV and HCV RNA.
1:45 HistoMosaic and Virtual Color Multiplexing Pave the Way to High-Throughput Analysis of Genetic Heterogeneity and Overexpression in Tissue Samples
Emil Kartalov, Ph.D., Assistant Professor, Pathology, Keck School of Medicine, University of Southern California
We present two novel interlocking techniques – HistoMosaic and virtual-color PCR multiplexing. HistoMosaic allows for high-throughput analysis of tissue samples by large-scale microfluidic compartmentalization and parallel PCR reactions. Virtual-color PCR multiplexing allows for dozens of questions to be posed to the same reaction volume while using the installed base of PCR machines. The integration of these techniques would produce a powerful new tool for cancer diagnostics and fundamental cancer research.
2:15 Sponsored Presentations (Opportunities Available)
2:45 A Method to Perform Selective DNA or RNA Extraction from Single Cells and Digital PCR in the Same Nano Wells
Hai-Qing Gong, Associate Professor, School of Mechanical and Aerospace Engineering, Nanyang Technological University
We developed a novel microfluidic technique to perform near-zero-loss nucleic acid extraction and purification from low copy cells (1-105 cfu) in a microwell and furthermore perform single cell realtime PCR in the same microwell. We further applied this "all-in-one-well" sample prep integrated PCR to an array of nanoliter wells for single cells dPCR analysis. This sample prep integrated dPCR method has a potential to carry out dPCR using a raw sample with zero loss of nucleic acid when removing PCR inhibitors. It also has benefits of reducing the total raw sample volume to 1ul or less, and selectively remove DNA or RNA from the raw sample.
3:15 Close of Conference
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